Glaucoma, a leading cause of blindness worldwide, damages the Retinal Nerve Fiber Layer (RNFL), which consists of retinal ganglion cell axons. Early diagnosis and treatment can prevent irreversible visual loss. Current optical methods used in clinical diagnosis of glaucoma are often limited to assessing RNFL thickness, which is insensitive to detect early glaucomatous damage. Critical barriers to effective early detection of glaucoma include 1) incomplete understanding of the relationship between RNFL optical properties and its underlying structures and 2) lack of knowledge of changes in the relationship in glaucoma. The goal of this proposal is to identify the relationship between RNFL optical properties and its underlying structures. F-actin, microtubules (MTs) and neurofilaments (NFs), the major cytoskeleton of axons, are the possible candidates for RNFL optical properties. In this project, we will 1) determine the role of F-actin in RNFL reflectance, 2) provide quantitative relationships between RNFL reflectance, birefringence and MTs and 3) test if change of RNFL reflectance speckle, an optical interference pattern, can differentiate between normal and glaucomatous retinas. Our central hypothesis is that ultrastructure change in early glaucoma manifests itself as changes in RNFL optical properties. We will test this hypothesis by determining the relationships among RNFL optical properties, axonal cytoskeleton and glaucomatous damage. Our long term goal is to provide sensitive assessment methods for early diagnosis of glaucoma and prevent irreversible visual loss. Aim 1: Hypothesis: axonal F-actin contributes to RNFL reflectance. We will determine F-actin's contribution to RNFL reflectance by depolymerizing F-actin with depolymerizing agents. Significance: Understanding the role of F-actin in RNFL reflectance enables us to assess the capability of RNFL reflectance measurement to detect F-actin alteration, an early sign of glaucoma damage. Aim 2: Hypothesis: change of RNFL reflectance and birefringence can provide a quantitative estimate of MT change in glaucoma. We will determine the relationships between RNFL reflectance, birefringence and MT density by measuring these properties and MT density of the same nerve fiber bundles. Significance: The obtained relationship will provide a direct estimate of MT change in glaucoma. Aim 3: Hypothesis: temporal change of RNFL reflectance speckle can differentiate between normal and glaucomatous retinas. Our recent study demonstrated that temporal change of RNFL speckle is associated with axonal dynamic activity. We will take a series of RNFL reflectance images and quantify RNFL speckle in normal and glaucomatous retinas. Significance: Because glaucomatous damage impairs axonal transport, this study is expected to demonstrate that change of RNFL speckle can detect change of axonal dynamic activity, a novel idea for detecting physiologic activity of axons with non-invasive optical methods. The knowledge gained can be used to design sensitive methods able to detect early glaucomatous damage.